The filoviruses, Ebola and Marburg viruses (EBOV and MARV), are NIAID category A viruses associated with outbreaks of severe viral hemorrhagic fever. FDA-approved drugs for prophylaxis or treatment of these infections are currently lacking. This project will develop small molecule antivirals targeting the filovirus VP35 protein, which carries out several functions, of which two are critical for efficient filovirus replication. First, VP35 inhibits the host interferon (IFN)-a/( response, a critical component of the host innate immune response to virus infection. Second, VP35 is required for viral polymerase function. The structures of the carboxy-terminal VP35 interferon inhibitory domain (IID), alone and in complex with dsRNA, have recently been solved. Analysis of these structures, combined with biochemical and functional studies, suggest that the VP35 polymerase co-factor and anti-IFN functions are carried out by two separate sets of basic residues in the IID. Interestingly, these two basic patches are linked by highly conserved hydrophobic residues and biochemical analyses reveal a very low tolerance for substitutions near these conserved functional elements. Analysis of recombinant VP35-mutant EBOVs has demonstrated a critical role for VP35 IFN-antagonist function for EBOV virulence. Together, these findings suggest that ZEBOV VP35, particularly the IID, is an ideal candidate for targeting by small molecules. Based on the high sequence conservation of the VP35 IID among filoviruses, such molecules are likely to effectively target multiple members of this virus family. A multidisciplinary team, with expertise in computational approaches to small molecule drug discovery, structural biology, biochemistry, signal transduction, virology, and BSL4 work, has been assembled to develop small molecule antivirals targeting VP35. The approach will first characterize, in detail, the VP35 protein as an antiviral target. To accomplish this, structural studies will be performed to identify conserved, functionally critical regions of VP35 and a combination of biophysical, biochemical, cell biology-based and virology-based assays will define sequence and structural features that are critical for VP35 function and are amenable to targeting with small molecules. Second, compounds will be developed to inhibit VP35 IFN-antagonist and/or polymerase co-factor functions. To accomplish this, an in silico screen will be performed to identify candidate compounds that interact with functionally critical domains within VP35. Compounds identified through this screen will be assessed for binding to VP35 by a battery of biochemical and biophysical studies in vitro. Compounds that score positive for VP35 binding will be subjected to structure-activity analyses for iterative optimization and in parallel subjected to structural analysis in order to produce compounds that inhibit, in the nanomolar-micromolar range, cell-based assays of VP35 IFN-antagonist and polymerase co-factor functions. Potent compounds will be characterized in terms for antiviral activity against EBOV and MARV. The completed studies will provide lead compounds for testing in in vivo models of EBOV pathogenesis.